Tirandamycin and process for making same

ABSTRACT

Antibiotic tirandamycin producible by culturing Streptomyces tirandis var. tirandis in an aqueous nutrient medium. Tirandamycin is active against Bacillus subtiles and can be used to minimize or prevent odor in fish and fish crates caused by this organism.

United States Patent Sebek et al. 1 June 20, 1972 s41 TIRANDAMYCIN ANDPROCESS FOR [56] References Cited I MAKING SAME OTHER PUBLICATIONS 72]inventors: Oldrich K. Sebek, Kalamazoo; Curtis E. Sugawara et al., .I.of Antibiotics. Vol. 10, set. A. 1957, pages Meyer, Galesburg, both ofMich. i38- I42.

[73] Assignee: The Upjohn Company, Kalamazoo, Mich. I Primary mminerkmme Goldberg [221 Filed: Sept. 8, 1969 Anomey-George T. .lohannesen andJohn Kekich Antibiotic tirandamycin producibie by culturingSrreplor'nyces il 424/221,; u-mndis van tirandis in an aqueous nutrientmedium Tiranda [58] Field of Search ..424/120; 195/80 and be usedminimize or prevent odor in fish and fish crates caused by thisorganism.

9 Claim, 1 Drawing Figure PKTENTEDJUHO m2 illilllll lllllll JlllllIllllJllll NOISSIWSNVHJ.

CURTIS E MEYER OLDRICH K. SEBEK ATTORNEY TIRANDAMYCIN AND PROCESS FORMAKING SAME BRIEF SUMMARY OF THE INVENTION Tirandamycin (U-29,275) is anamphoteric phenolic compound which is producible by culturing atirandamycinproducing actinomycete in an aqueous nutrient medium. It hasthe property of adversely affecting the growth of Grampositive bacteria,for example, Staphylococcus aureus, Streptococcus hemolyticus,Streptococcus faecalis, Bacillus subtilis, and Diplococcus pneumoniac.Accordingly, tirandamycin can be used alone or in combination with otherantibiotic agents to prevent the growth of or reduce the number ofbacteria, as disclosed above, in various environments.

DETAILED DESCRIPTION OF THE INVENTION CHARACTERIZATION OF TIRANDAMYCINAS THE SODIUM SALT Elemental Analyses Calcd. for C H,,NO-,Na:

C, 60.1 I; H, 5.96; 0, 25.55; N, 3.18; Na, 5.24

Found:

C, 58.1 I; H, 6.28; N, 2,86; Na, 4.74 Ultraviolet Spectrum Tirandamycinsodium salt has a characteristic ultraviolet absorption spectrum asfollows:

The infrared absorption spectrum of tirandamycin sodium salt suspendedin mineral oil mull is reproduced in the drawing. Tirandamycin showspeaks at the following wave lengths expressed in reciprocal centimeters:

Band Frequency Band Frequency (cm") Intensity (cm') Intensity 3720 MI210 S 3600 M I155 S 3360 S 1130 S 3300 sh S I120 S 3130 M I087 S 3000(oil) S I068 S 2960 (oil) S 1055 S 2900 (oil) S 1045 M 2760 W I020 S1730 S I000 M I670 S 995 M I640 S 960 M I625" l 590 S 955 M (Broad) I530S 930 W I485 S 920 W 1470 (oil) S 907 M 1415 S 900 M 1395 S 893 M I383 S865 S I355 (oil) M 840 W I345 M 8I5 W I320 M 805 W I305 M 795 M I295 M780 W I290 M 763 M I260 S 745 S I255 S 725 S Band intensifies areindicated as S"M", and W", respectively, and are approximated in termsof the backgrounds in the vicinity of the bands. An S band is of thesame order of intensity as the strongest in the spectrum; M bands arebetween one-third and two-thirds as intense as the strongest band, and Wbands are less than one-third as intense as the strongest band. Theseestimates are made on the basis of a percent transmission scale. Thedesignation SH" denotes a shoulder.

Ferric chloride test Positive Molish Negative Ninhydrin SakaguchiNegative Millon Biuret Negative Solubilities Soluble in moist chloroformand in ethylene chloride.

CHARACTERIZATION OF TIRANDAMYCIN AS THE BROMOBENZENE SOLVATE ElementalAnalyses Calcd for: C H NqBrz C, 58.49; H, 5.61; N, 2.43; O, 19.49; Br,13.9l Found: C, 58.68; H, 5.44; N, 2,35; 0, 19.92; Br, 13.25

Molecular Weight High resolution mass spectrometry gave a measured massof 417.1792 which corresponds to C l-ENC having a theoretical massof417.1786.

Tirandamycin as the amorphous free phenol is very soluble in diethylether, ethyl acetate, acetone (and does not seem to solvate in acetonewhereas sodium salt gives solvate insoluble in acetone). It is alsosoluble in chloroform, ethylene chloride, and lower alcohols, forexample, methanol and ethanol. The amorphous free phenol of tirandamycinis insoluable in water and petroleum ether. It is transitorily quitesoluble in benzene but rapidly forms a solvate which crystallizestherefrom.

THE ANTIBACTERIAL PROPERTIES OF TIRANDAMYCIN Minimum Inhibitory TestOrganism Concentration (NS/ml.) S taphylococcur aureu: 250 Streptococcushemolyticus 0.5 Streptococcus faecalis 0.5 Bacillus .subtilis 8Diplococcus pneumoniae 0.5

THE MICROORGANISM The actinomycete used according to this invention forthe production of tirandamycin is Streptomyces tirandis var. tirandis.One of its strain characteristics is the production of tirandamycin. Asub-culture of the living organism was deposited without restriction andcan be'obtained from the permanent collection of the NorthernUtilization and Research Division, Agricultural Research Service, US.Department of Agriculture, Peoria, Illinois, USA. Its accession numberin this repository is NRRL 3689.

(1962)]Microscopic Characteristics 3 The microorganism of this inventionwas studied and characterized by Alma Diet: of the Upjohn ResearchLaboratories.

DESCRIPTION OF THE MICROORGANISM sections of Pridharn et al. [AppliedMicrobiol.6:52-79 Tlm l t me!.hseeuasdwithenxet tbs. is s ted. in he e.ssstio s. on. th ba is. 92.2 3219: teristics noted in the literaturecitations or by actual comparison as was possible for most of theorganisms listed.

The new organism might be placed in the Aureus, Dia-' stations, orAntibioticus Series of Baldacci [Giomale di Microbiologia 6:10-27(1958)] or the Cinereus-color group of l-liitter [Systematik derStreptomyceten unter bcsondere Bs i qksish i uasdsr. vqmihnes .sb. ldsn...AL ib iL-.L l(arger,.Basel (1967)]. However, the singlecharacteristic of aerialcolor mass is insufficient to relate the neworganism to the cultures included in these color groups. The new culturediffers significantlyin pigment characteristics, growth oncarboncompounds in synthetic media and antibiotic production fromorganisms assigned to those color groups. I S. tirandishad somesimilarity to Streptomyces melanogenes [The Journal of Antibiotics, Ser.A 10:138-142 (1957)] on Ektachrome. S. melanogenes was differentiated bytrace gray aerial growthon Oil percent tyrosine agar and casein starchagar and red-tan and yellow-tan reverses on these media. S. inelanagenesdid not solubilize casein but did solubilize xanthine. It also producesthe antitumor antibiotic melanomycrn. I

S. rirandis can be differentiated from named species in the Upjohnculture collectionand from those described in the literature. It isreadily identified by its color pattern, its sporophore and spore typeand antibiotic production pattern. Therefore, it is proposed that thenew soil isolate be designated Streptomyces n'randis Dietz sp. n. andthat this type species be designated the type variety Srrepromycestirandis var. tirandis in accordance with Rule 911(2) of thelntemational Code of Nomenclature of Bacteria llntem. J. System.

Bacteriol. l6 :459490 1966)]. Srreptomyces u'randis Dietz sp. n. ColorCharacteristics Aerial growth gray (gray-white, gray-cream, gray-tan).Melanin positive. Appearance on Ektachrome [Ann. NY.

Acad. Sci. 60:152-154 (1954)]is given in Table 1. Reference colorcharacteristics are given in Table 2. The culture may be placed in theWhite (W), Yellow (Y), and Gray (G) color series of Tresner and Backus[Applied Microbioi. 11:335-338 Sporophores small, straight, to openspiral to spiral (RF, RA, 8) in the sense of Pridham et al. (supra).Spores smooth, long, many angled at ends (appearing octangular) bydirect electron microscopic examination. Spores ridged with muchZsurt'a-ce detail when examined by the carbon replication 'method of IDiet: and Mathews [Applied Microbiol. 10:258-263 (1962); 16:935-941(1968)].

Cultural and Biochemical Characteristics I See Table 3.

Carbon Utilization Growth of the culture on carbon compounds wasdeterminedin' the synthetic medium of Pridham and Gottlieb [J Bacteriol.56:107-114 (1948)] and in their modified medium [International Journalof Systemic Bacteriology 16:313-340 (1966)]. .In the former, the cultureshowed slightgr gwth on the control; good growth on D-xylose, L-arabinose, rhamnose, D-frustose, D-galactose, D-glucose, D-mannose, maltose,sucrose, lactose, cellobiose, raffinose, dextrin, inulin, solublestarch, glycerol, D-mannitol, D-sorbitol, inositol, and sodium acetate;moderate growth on salicin, sodium oxalate, sodium tartrate, sodiumcitrate, sodium succinate; slight growth on dulcitol, phenol, andcresol. There was no growth on sodium salicylate. Inv the modifiedmedium there was no growth on the negative control. There was goodgrowth on the positive control (glucose). Growth was equal to or betterthan on the glucose control on L-arabinose, sucrose, D-xylose, inositol,D- mannitol, D-fructose; rhamnose, and ratfinose. Growth was doubtful oncellulose. Temperature Growth was fair at 1 8 C., good at 24-37 C. onBennetts and maltose-tryptone agars. 0n Czapeks sucrose agar, growth wasfair at 18 C. and slight at 24-37 C. On all media there was tracevegetative growth in 24 hours at 45 C. and C.

The characteristics of Streptomyces tirandis Dietz sp. n.,

NRRL 3689, are given in the following tables:

Table 1 Appearance of S. tirandis on Ektachrome Table 2 Reference ColorCharacteristics of S. lirandis Table 3 Cultural and BiochemicalCharacteristics of S. tirandis Appearance of S. tirandis on Ektachrome'Agar Medium Surface I Reverse Bennett's Gray Brown Czapek'a sucroseTrace gray Colorless Maltoae-tryptone Trace gray Brown Peptone-iron Noaerial growth Brown 0.1% tyrosine Fair gray Brown Casein starch Fairgray Tan-brown 'Dietz, A., Ektachrome Transparencies as Aids inActinomycete Classification," Annals of the New York Academy ofSciences, Q!l-.l$1l2-....

.. TABLE 2 Reference Color Characteristics of S. iirandis ISCC-NBSmethod of designating color and a dictionary of color names,

Agar medium Color harmony manual 3rd ed.,'1948 Z circular 553 a S 301) 1sand a 91gm dark grayish yellow. Bennett's R 210 light mustard tan ggglllghlttolilve brown.

- g g 0 we. P 2ge covert tan, gr'lege .{94m light olive brown.

. 109gm light grayish olive.

' S 2cb ivory tlnt- 92m yellowish white. v 93gm yellowish gray.'Czapek's sucrose R 1 5 ec putty gggm grahyish lyellow. Y gm ye OWlSgray. P S 3il1 beige gray, mouse; oliraii gray.

* m 111 um gra Maltose-tryptone R Spl clove brown, deep brownuiggmdmiiierzife yelgotwish brown.

, g er ye owrs rown. 5 gig ifiiobe brown, cinnamon brown, light browngg'm giggeato yelllpwish brown.

es ver gray gm g rowms gray. 1 R 3ie camel, maple sugar, tan to, "ggmliggt ytellowlilsh brlrlwgn. t

gmo eraeye OWlS rown 0.... Yeast extract malt .extract (ISP 2) 41g lightspice brown, sandalwood, toast tan lighi'abrogvrig gm mo era e rown. P

3ge beige, camel {79m light grayish yellowish brown.

TABLE 2-Continucd Agar medium Color harmony manual 3rd ed., 1048 2 S 3fesilver gray ti3gm light brownish 'ra Oatmeal (ISP-3) .{R c biscuit,ectu, oatmeal. sand.. DOgm grayish yellow. 3

l" Zea light ivory, eggshell.. Xllgni yellow pale. is lde natural,string. h3gni yellowish gray. Inorganic-salts starch USP-4) R 31g adobebrowmcinnamon brown, light brown TTgni moderate yellowish ln'own.

l 2ge covert tan, grlege H ilOgm grayish yellow. s 3el) sand I fllgnldark grayish yellow. Glycerol-asparagine USP-5) R 2ie light mustard tan..{'.Hg light olive hrown.

P 106g light olive.

NoTE.S=Surfaee. P=Pig ment. R=Reverse.

1 Matte surface used for all chips.

1 Jacobson, E., \\.C. Granville, and C.E. Foss. 1048. Color IlarmouyManual, 3rd Ed. (ontainer (.orporation of America. (.hieago. lllinois. 3Kelly, l-..L.. and D.B. Judd. 1955. The ISCC-NBS Method of DesignatingColors and a Dictionary of Color Names. Dept. (.onnn. Fire. 553.

TABLE 3 ultural and Biochemical Charaeteristies of S. tirandis Surfacediu Reverse Other Agar media:

leptone-iroiL. No aerial growth to very slight trace gray aerial Brown..llrowu pigment.

growth.

Calcium iiialate.'.. Gray-cream to gray-tan aerial growth (lray .[Nopigment.

(Malate not solnhilized. Glueose-asparagine (tray-cream aerial growth.Yellow-olive No pigment. Skim milk Trace to good gray-cream aerialgrowth Yellow-tan. l Yellow-tan pigment.

(Casein soluhilized around growth. Tyrosine Gray-cream aerial growth.Brown Brown pigment.

Tyrosine soluhilized.

Xanthiue Gray aerial growth. Yellow lale yellmr-tan pigment.

'lXanthine not soluhilized. Nutrient starch Gray-white aerial growth.Yellow. .j'Yt'llOW pigment.

[Stareh hydrolyzed. Yeast extract-malt extract Gray-cream aerial growthYellow-tan. Yellow-tan pigment. Beuuetts Good gray aerial growth.(lream-tan.... lale yellow pigment. (zapek's sum-050.. Traee gray-whit eaerial growth.. Gray. No pigment. .\laltose-tryptone. (lood gray aerialgrowth. Tan. lale tan pigment. leptone-yeast extract-iron USP-6).. .\'oaerial growth Brown. Brown. Tyrosine (ISP-T) (ream-gray aerial growth.Brown.. .i Very slight traee brown pigment.

(i elatin media:

Traee gray aerial llaiu Traee gray aerial growth... Nutrient Brothmedia:

Synthetic nitrate Nutrient nitrate Litmus milk Trace gray aerial growthon brown surface ring Traee white. to gray aerial growth on surface ring.lT-an pigment in upper of medium.

lLiquef-aetion complete. lTan pigment in upper of medium. (Liquefactioncomplete.

.lFloceulent bottom growth.

[Nitrate redueed to nitrite. {Tan pigment.

. Floeeulent bottom growth.

N'trate reduced to nitrite.

plI 6.2-7.2.

The new compound of the invention is produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. lt is to be understood also that for the preparation oflimited amounts surface cultures and bottles can be employed. Theorganism is grown in a nutrient medium containing a carbon source, forexample, an assimilable carbohydrate, and a nitrogen source, forexample, an assimilable nitrogen compound or proteinaceous material.Preferred carbon sources include glucose, brown sugar, sucrose,glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like.Preferred nitrogen sources include com steep liquor, yeast, autolyzedbrewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal,milk solids, pancreatic digest of casein, distillers' solids, animalpeptonc liquors, meat and bone scraps, and the like. Combinations ofthese carbon and nitrogen sources can be used advantageously. Tracemetals, for example, zinc, magnesium, manganese, cobalt, iron, and thelike, need not be added to the fermentation media since tap water andunpurified ingredients are used as media components. 7

Production of the compound of the invention can be effected at anytemperature conducive to satisfactory growth of the microorganism, forexample, between about 18 and C., and preferably between about 20 and 32C. Ordinarily, optimum production of the compound is obtained in about 2to ID days. The medium normally remains basic during the fers present,if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganism for inoculation to avoid a pronounced lag in theproduction of the new compound and the attendant inefficient utilizationof the equipment. Accordingly, it is desirable to produce a vegetativeinoculum in a nutrient broth culture by inoculating this broth culturewith an aliquot from a soil or a slant culture. When a young, activevegetative inoculum has thus been secured, it is transferred asepticallyto large vessels or tanks. The medium in which the vegetative inoculumis produced can be the same as, or different from, that utilized for theproduction of the new comound, as long as it is such that a good growthof the microorganism is obtained.

The new compound of the invention is an amphoteric chemical compound. Inthe form of the free phenol it is very soluble in diethyl ether, ethylacetate, acetone, chloroform, ethylene chloride, methanol, and ethanol.It is relatively insoluble in water and petroleum ether. Tirandamycin istransitorily quite soluble in benzene but rapidly fonns a solvate whichcrystallizes therefrom. In this regard, tirandamycin as the sodium saltreadily forms a solvate with acetone, and in the free phenol form formssolvates with benzene and bromobenzene. These solvates are stablecompounds and usefermentation. The final pH is dependent, in part, onthe bufful to characterize the free phenol form of tirandamycin.

nan

lOlMl mm A variety of procedures can be employed in the isolation andpurification of tirandamycin, for example, solvent extraction, partitionchromatography, silica gel chromatography, liquid-liquid distribution ina Craig apparatus, and crystallization from solvents. Solvent extractionprocedures are preferred for commercial recovery inasmuch as they areless time consuming and less expensive.

In a preferred recovery process, tirandamycin is recovered from itsculture medium by separation of the mycelia and undissolved solids byconventional means, such as by filtration or centrifugation. Theantibiotic is then removed from the filtered or centrifuged broth byextraction. For the extraction of tirandamycin from the filter broth,water-immiscible organic solvents in which it is soluble, for example,l-butanol, methyl ethyl ketone, benzene, and methylene chloride(preferred) can be used. Advantageously, the extraction is carried onafter the filtered beer is adjusted to a pH of about 2 to 4 with amineral acid. When the filtered beer is extracted at a pH of 6 or above,the base salt, for example sodium, is extracted by chlorinatedhydrocarbons, for example, methylene chloride. When such extracts areconcentrated, the solvated crystalline salt of the antibiotic can beprecipitated with solvents such as acetone, benzene, or bromobenzene.

The extract obtained from an extraction of the filtered beer withmethylene chloride at a pH of about 2 to 4 can be converted to a salt byadjusting the pH to a basic pH with a base, for example, sodiumhydroxide, and the preparation freeze dried. This preparation can beused in environments where higher purity of the antibiotic is notessential.

Tirandamycin can be purified from a crude preparation, as describedabove, by subjecting the preparation to chromatography on a silica gelcolumn which is developed with a solvent system consisting of 5%methanol in methylene chloride. Active fractions, as determined bybioactivity against the microorganism B. cereus, the assay which isdescribed infra, are pooled and concentrated to dryness.

Also, tirandamycin can be purified by successive transfers fromprotonated to non-protonated forms and vice versa, especially with othertypes of treatments intervening as, for example, solvent extractions andwashings, chromatography, and fractional liquidliquid extraction. Inthis manner, salts of tirandamycin can be employed to isolate or upgradethe antibiotic.

Tirandamycin forms salts with alkaline metals, alkaline earth metals,and amines. Metal salts can be prepared by dissolving tirandamycin inmethanol, adding a dilute metal base until the pH of the solution isabout 7 to 8, and freeze drying the solution to provide a dried residueconsisting of the tirandamycin metal salt. Tirandamycin metal saltsinclude the sodium, potassium, and calcium salts. Amine salts oftirandamycin, including those with organic bases, such as primary,secondary, and tertiary, mono-, di-, and polyamines also can be formedusing the above-described rather commonly employed procedures. Othersalts are obtained with therapeutically efiective bases which impartadditional therapeutic effects thereto. Such bases are, for example, thepurine bases such as theophyllin, theobromin, caffeine, or derivativesof such purine bases; antihistaminic bases which are capable of formingsalts with weak acids; pyridine compounds such as nicotinic acid amide,isonicotinic acid hydrazide, and the like; phenylalkylamines such asadrenaline, ephedrine, and the like; choline, and others.

Tirandamycin, its salts and solvates are active against Bacillussubtilis and can be used for treating breeding places of silk worms toprevent or minimize infections caused by this organism. The novelcompounds of the invention also are active against Streptococcusfaecalis and can be used to disinfect washed and stacked food utensilscontaminated with this bacteria.

Hereinafier are described non-limiting examples of the process andproducts of the present invention. All percentages are by weight and allsolvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Part A Fermentation A soil stock of Streptomyces tirandis var.tirandis NRRL 3689, is used to inoculate a series of 500-ml. Erlenmeyerflasks, each containing ml. of sterile preseed medium consisting of thefollowing ingredients:

Glucose monohydrate 25 g.lliter Pharmarnedia' 25 g.lliter Tap water q.s.Balance 'Pharmamedia is an industrial grade of cottonseed flour producedby Trader's Oil Mill Company, Fort Worth, Texas.

The flasks are grown for 3 days at 28 C. on a reciprocating shaker.

Preseed inoculum, described above, is used to inoculate a 20-liter seedtank containing sterile medium consisting of the Wilson's Peptone LiquorNo. 159 is a preparation of hydrolyzed proteins of animal origin.

The seed I tank is inoculated with 5 percent (volume/volume) of thepreseed inoculum. The seed tank is incubated at 28 C. for 2 days withagitation at the rate of 400 rpm and aeration at the rate of 10 standardliters/minute. The medium is adjusted to pH 7.2 with aqueous sodiumhydroxide before sterilization.

The seed inoculum, described above, is used to inoculate a 400-literfermentation tank containing 250 liters of sterile fermentation mediumconsisting of the following ingredients:

Bacto Peptone (Difco) 5 g.lliter Sodium chloride 5 g.lliter CornsteepLiquor 0.1 g.lliter Tap water Balance The fermentation medium isinoculated with 5 percent (volume/volume) of the seed inoculum,described above. The fermentation proceeds for 5 days during which timethe fermentation medium is agitated at a rate of 280 rpm, and aerationprovided at the rate of 200 liters/minute. The temperature in thefermentation tank is maintained at 28 C. When foaming occurs, lard oilis used as antifoam.

A typical tirandamycin fermentation, as described above, can beillustrated by the following assay profile:

Assay Hours (biounits/ml.) 0 69 l 1.4 92 16.0 1 l4 1 2.6

The assay is a disc plate assay using the microorganism Bacillus cereus.The assay is conducted at a pH of 7.0. A standardized spore suspensionof B. cereus (0.5 ml.) is inoculated into 1 liter of melted BBL seedagar (30.5 g.lliter), supplied by Baltimore Biological Laboratories.Agar plates are then poured, lfi-inch' paper discs saturated withtesting material are placed on the agar, and the plates are incubatedovernight at 32 C.

One biounit (BU) is defined as that amount of the active material whichgives a 20 mm. diameter zone of inhibition. 1 Mg. (1,000 mcg.) oftirandamycin assays approximately 270 BU. Thus, a preparation assaying4.0 mcg. tirandamycin per ml. is converted to 1.08 BU.

Par B-Recovery Whole fermentation beer I ,000 ml. assaying 1.08 BUagainst B. cereus), prepared as described above, is filtered with theaid of about percent diatomaceous earth. The filter cake is washed withl/ l 0 beer-volume of water. The combined clear beer and wash isadjusted to pH 2.0 with sulfuric acid and then extracted with one-halfvolume of methylene chloride. The methylene chloride extract isconcentrated in vacuo. Additional water is added and the remainder ofthe methylene chloride is removed by distillation. The remaining aqueoussuspension is adjusted to a pH of 7.5 with 50 percent aqueous sodiumhydroxide and freeze dried; yield, 62 mg. of crude tirandamycin assaying34.0 mcg./mg. against B. cereus. Part C--Purification To 750 ml. of amethylene chloride concentrate, obtained as described above in Part B,is added 2 liters of water, and while the mixture is stirred, the pH israised to with 5 N sodium hydroxide. The aqueous extract isback-extracted into 500 ml. of methylene chloride at a pH of 6.3. Afterdrying and concentrating the extract, it is chromatographed on a silicagel column developed with 5% methanol in methylene chloride. Fractionsfrom the column which are bioactive against B. cereus are pooled andconcentrated to dryness in vacuo. The dried solid is dissolved inchloroform and 5 percent methanol is added. Addition of more methanoland refrigeration results in crystallization of the sodium salt oftirandamycin solvated with chloroform. A portion of this sodium salt oftirandamycin is converted to the free phenol by dissolving l g. in 200ml. methylene chloride and ml. methanol. The pH is adjusted to 2.0 with4.5 N alcoholic hydrogen chloride. The solution is washed free of sodiumchloride, dried with anhydrous sodium sulfate, and concentrated todryness in vacuo; yield, 800 mg. of tirandamycin in the free phenolform.

EXAMPLE 2 By reacting tirandamycin sodium salt, as prepared in Examplel, with acetone, there is obtained the acetone solvate of tirandamycinsodium salt. This compound is active against various Gram-positiviebacteria.

EXAMPLE 3 By reacting tirandamycin free phenol form, as prepared inExample 1, with bromobenzene, there is obtained the bromobenzene solvateof tirandamycin. This compound is active against various Gram-positivebacteria.

EXAMPLE 4 By substituting benzene for bromobenzene in Example 3 there isobtained the benzene solvate of tirandamycin. This compound is activeagainst various Gram-positive bacteria.

We claim:

1. A composition of matter selected from the group consisting oftirandamycin, a compound which a. is effective in inhibiting the growthof various Gram-positive bacteria; and, as the sodium salt;

' ing Streptomyces tirandis var. tirandis in an aqueous nutrient b. hasan optical rotation [01],, +5 1 (c, 1 percent in d. has a characteristicinfrared absorption spectrum as shown in the accompanying drawing, andas the bromobenzene solvate;

e. has the following calculated empirical formula and elementalanalyses: C H NO-Br C, 58.68; H, 5.44; N, 2.35; O, 19.92; Br, 13.25; and

f. has a measured mass of 417.1792 as determined by high resolution massspectrometry; and non-toxic base addition salts thereof.

2. Composition of matter in dry form comprising tirandamycin, thecompound defined in claim 1, said composition of matter assaying about34.0 meg/mg. of tirandamycin on the B. cereus assay.

3. The compound, tirandamycin, according to claim 1, in its essentiallypure free phenol form.

4. Sodium salt of tirandamycin according to claim 1.

5. The compound, according to claim 4, in its essentially purecrystalline form.

6. The bromobenzene solvate of tirandamycin, the compound defined inclaim 1.

7. A process for making tirandamycin, the compound defined in claim 1,which comprises cultivating Streptomyces tirandis var. tirandis in anaqueous nutrient medium under aerobic conditions until substantialantibiotic activity is imparted to said medium by the production oftirandamycin.

8. A process according to claim 7 which comprises cultivatmediumcontaining a source of assimilable carbohydrate and assimilable nitrogenunder aerobic conditions until substantial antibiotic activity isimparted to said medium by the production of tirandamycin and isolatingthe tirandamycin so produced.

9. A process according to claim 8 in which the isolation comprisesfiltering the fermentation medium, extracting the filtrate with awater-immiscible solvent for tirandamycin, and recovering tirandamycinfrom the solvent extract.

nines no-

2. Composition of matter in dry form comprising tirandamycin, thecompound defined in claim 1, said composition of matter assaying about34.0 mcg./mg. of tirandamycin on the B. cereus assay.
 3. The compound,tirandamycin, according to claim 1, in its essentially pure free phenolform.
 4. Sodium salt of tirandamycin according to claim
 1. 5. Thecompound, according to claim 4, in its essentially pure crystallineform.
 6. The bromobenzene solvate of tirandamycin, the compound definedin claim
 1. 7. A process for making tirandamycin, the compound definedin claim 1, which comprises cultivating Streptomyces tirandis var.tirandis in an aqueous nutrient medium under aerobic conditions untilsubstantial antibiotic activity is imparted to said medium by theproduction of tirandamycin.
 8. A process according to claim 7 whichcomprises cultivating Streptomyces tirandis var. tirandis in an aqueousnutrient medium containing a source of assimilable carbohydrate andassimilable nitrogen under aerobic conditions until substantialantibiotic activity is imparted to said medium by the production oftirandamycin and isolating the tirandamycin so produced.
 9. A processaccording to claim 8 in which the isolation comprises filtering thefermentation medium, extracting the filtrate with a water-immisciblesolvent for tirandamycin, and recovering tirandamycin from the solventextract.